This invention relates to the production of highly purified plasmid DNA, and in particular to production and isolation of pharmaceutical grade plasmid DNA for use in gene therapy.
A variety of methods are available for isolating and purifying plasmid DNA. In general, these methods take advantage of the physical differences between chromosomal DNA and plasmid DNA. In terms of size, chromosomal DNA is larger than plasmid DNA. When cells are lysed, the larger chromosomal DNA becomes linearized and entangled in the cellular debris and may be separated from the cell lysate.
Prior art methods of isolating and purifying plasmid DNA include lysis by boiling (Holmes and Quigley, Anal. Biochem. 114, 193 (1981)), lysis with alkali (Birnboim and Doly, Nuc. Acids Res. 7, 1513 (1979)), and lysis with detergent (Godson and Vapnek, Biochem. Biophys. Acta 299, 516 (1973)). PCT publication no. 95/21250 discloses a method of isolating plasmid DNA using detergent in combination with alkali treatment. Prior art methods also use highly toxic chemicals to extract and isolate the plasmid DNA, such as ethidium bromide, cesium chloride, phenol, and chloroform. Moreover, these methods are most effective with smaller plasmids; e.g. plasmids less than approximately 8-10 kb. As plasmid size increases, plasmid DNA isolation becomes more difficult using the existing prior art methods.
In general, methods that employ alkali quickly add a standard amount of sodium hydroxide to the cellular suspension (Birnboim and Doly, supra). The pH of the resulting solution rises rapidly, in some cases, to over 13, which results in much degradation of the plasmid DNA. In addition, the concentration of the cells in suspension is dilute (i.e., of the order of an optical density (OD) of 1-3 units at a wavelength 600 nm for a 1 cm light path) to maximize recovery of plasmid DNk
An object of the invention is to provide a method of preparing pharmaceutical grade DNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host RNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bactenal host protein.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host chromosomal DNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host endotoxins.
Another object of the invention is to provide a method of isolating and purifying relatively large plasmid DNAs.
Another object of the invention is to provide a scalable method of isolating large amounts of plasmid DNA in sufficiently pure form for use in gene therapy.
Another object of the invention is to maximize the yield of plasmid DNA from a host cell/plasmid DNA combination.
The invention is based on the discovery of a method for producing and isolating highly purified plasmid DNA. The plasmid DNA produced and isolated by the method of the invention contains very low levels of contaminating chromosomal DNA, RNA, protein, and endotoxins. The plasmid DNA produced according to the invention is of sufficient purity for use in vivo or ex vivo gene therapy.
Thus, the invention encompasses a process for producing and isolating highly purified plasmid DNA that includes the step of alkaline lysis in which the pH of the solution is monitored and raised in a controlled manner such that it does not rise above a predetermined pH value which is equal to 0.1 pH units below the irreversible alkaline denaturation value of the plasmid DNA.
Preferably, the predetermined pH value is equal to 0.2 pH units below the irreversible alkaline denaturation value of the plasmid DNA which is being isolated.
The predetermined pH value is within the range of 8.0 to 14.0 and is preferably within the range of about 81.0 to 13.1 and most preferably about 12.1 to 12.9.
The controlled rise in pH according to the method of the invention results in exceedingly low degradation of plasmid DNA, and thus permits higher yield of plasmid DNA.
As used herein, the terms xe2x80x9cdenaturexe2x80x9d, xe2x80x9cdenatured DNAxe2x80x9d and xe2x80x9cdenaturationxe2x80x9d are defined as conformations of DNA in which the hydrogen bonds between strands of double stranded DNA are ruptured. The term xe2x80x9cirreversible alkaline denaturation valuexe2x80x9d is defined as the pH value at which no more than about 50% of the alkaline denatured plasmid DNA fails to renature as determined by standard agarose gel electrophoresis. Determination of the irreversible alkaline denaturation value is described hereinbelow.
According to the invention, the alkaline lysis step is performed on cells harvested from a fermentation which has been grown to a biomass of cells that have not yet reached stationary phase, and are thus in exponential growth, about 2-10 g dry weight/liter.
In a preferred embodiment, the alkaline lysis step is performed on cells harvested from a fermentation which has been grown to a high biomass of cells that have not yet reached stationary phase and are thus in exponential growth.
As used herein, a xe2x80x9chigh biomassxe2x80x9d or xe2x80x9chigh densityxe2x80x9d is defined as a cellular concentration of approximately 10-200 g dry weight per liter, and preferably 12-60 g dry weight per liter. As used herein, the term xe2x80x9cexponential growthxe2x80x9d refers to that portion of the cellular growth cycle between the lag phase and the stationary phase when cells are doubling at a logarithmic rate.
The term xe2x80x9cexponential growthxe2x80x9d is also meant to encompass the late lag phase (i.e., the early stationary phase) which occurs between the logarithmic growth phase and stationary phase, when the cell growth rate is slowing, and therefor encompasses an extended exponential growth phase. Therefore, xe2x80x9cstationary phasexe2x80x9d refers to horizontal growth, i.e., when the cells have essentially stopped dividing and have reached a quiescent stage with respect to cell doubling. According to the invention, the combination of controlled pH increase and lysis at high cell density from cells harvested during exponential growth produces a high yield of intact and highly pure plasmid DNA from a single batch of cells.
The invention also encompasses a method for determining the optimum lysis conditions for lysing host cells containing plasmid DNA, comprising the steps of a) growing a culture of bacterial host cells to a cell density within the range of about 12 g to about 60 g per liter dry weight units; b) lysing the bacterial cells during exponential growth at a pH of said culture sufficient to cause cell lysis and to cause denaturation of no greater than 50% of plasmid DNA contained in said cells; and c) selecting a pH value for optimum lysis conditions which is about 0.1 pH units below the pH of step b).
Preferably, the lysing step b) is performed at a pH sufficient to cause denaturation of no greater than 90-95% of plasmid DNA, and the pH selected in step c) is about 0. 17-2.0 pH units below step b) pH.
Determining an optimum lysis pH or an optimum sodium hydroxide concentration for cell lysis according to the invention permits a longer time period during which cell lysis may occur, which in turn allows for a) destruction of larger amounts of undesirable endotoxin which may be present in the plasmid DNA preparation, b) denaturation of larger amounts of chromosomal DNA, and c) precipitation of larger amounts of chromosomal DNA, without concommitant loss of plasmid DNA in terms of quantity or loss of high quality plasmid DNA produced according to the invention.
The invention also thus encompasses a fermentation process which maximnizes yield of plasmid DNAs from large scale cultures of transformed host cells. The fermentation process includes controlling the growth rate such that the supply of metabolites essential for growth is adequate to permit growth to a high biomass, but is not in excess so as to inhibit such growth. It is critical to this aspect of the invention that growth is not reduced by supplying inhibitory concentrations of metabolites and catabolites. However, it is also critical that components necessary for plasmid DNA production, such as nucleotides or nucleotide precursors, are not limiting during the fermentation process.
Another aspect of the invention is that plasmid DNA yield and quality is not reduced by insufficient concentrations of metabolites and nucleic acid precursors. Therefore, in another aspect the invention encompasses providing a culture growth medium having excess quantities of metabolites and nucleic acid precursors during the fermentation process; that is, concentrations in excess of those used in the prior art for the production of plasmid DNA in large scale fermentation.
In a preferred embodiment, the quantity of yeast extract in the batch medium is about 20 gm/liter, a 4-fold increase over conventional concentrations of yeast extract. In another preferred embodiment, the medium is supplemented with a source of organic nitrogen. Preferably, organic nitrogen is added to the culture medium in the form of ammonium salts such as. ammonium sulfate or ammonium nitrate, at about 5 gm/liter or 10 gm/liter, or ammonium phosphate at 3 gm/liter, 5 gm/liter or 10 gm/liter.
The invention further encompasses a method of producing and isolating highly purified plasmid DNA that is essentially free of contaminants and thus is pharmaceutical grade DNA.
A plasmid DNA preparation isolated according to the methods of the invention may be subject to purification steps including ion exchange chromatography which may include both fluidized bed ion exchange chromatography and axial and/or radial high resolution anion exchange chromatography, and further may include gel permeation chromatography.
These methods thus include the alkaline lysis step described herein in combination with subsequent ion exchange chromatography and gel permeation chromatography steps.
Alternatively, in a preferred embodiment of the invention, it has been discovered that high resolution anion exchange chromatography is not necessary to arrive at highly pure plasmid DNA. Therefore, the alkaline lysis step may be combined with a plasmid DNA isolation step which includes only fluidized bed ion exchange chromatography and does not include additional axial or radial high resolution anion exchange chromatgraphy. Therefore, in this method of the invention, the ion exchange chromatography step consists essentially of fluidized bed ion exchange chromatography. In this aspect of the invention, the method may further consist essentially of gel permeation chromatography.
Ion exchange chromatography and gel permeation chromatography facilitate rapid and large scale isolation of plasmid DNA, and allow one of skill in the art to avoid use of highly toxic chemicals such as ethidium bromide, chloroform, and phenol, at least traces of which often appear in the final preparation. According to the invention, the gel permeation chromatography step in the process also provides for isolation of the plasmid DNA in a pharmaceutically acceptable solution.
The terms xe2x80x9cessentially freexe2x80x9d, and xe2x80x9chighly purifiedxe2x80x9d are defined as about 95% and preferably greater than 98.99% pure or free of contaminants, or possessing less than 5%, and preferably less than 1-2% contaminants. xe2x80x9cPharmaceutical grade DNAxe2x80x9d is defined herein as a DNA preparation that contains no more than about 5%, and preferably no more than about 1-2% of cellular components, such as cell membranes, chromosomal DNA (preferably  less than 1%), RNA (preferably  less than 0.2%), protein (preferably  less than 1%) and other cell derived contaminants. Pharmaceutical grade DNA should contain no more than 100 EU/mg endotoxins, and is preferably predominant circular in form.
The invention also encompasses isolation of relatively large plasmid DNAs, i.e., in the size range of from approximately 5 kb to approximately 50 kb, preferably 15 kb to 50 kb, which DNA includes a vector backbone of approximately 3 kb, a therapeutic gene and associated regulatory sequences. Preferably, the vector backbone used in the method of the invention specifies a high copy number, a polylinker for insertion of a therapeutic gene, a gene encoding a selectable marker, e.g., the tetracycline or kanamycin resistance gene, and is physically small and stable. The approximate 3 kb vector backbone of the plasmid advantageously permits inserts of large fragments of mammalian, other eukaryotic, prokaryotic or viral DNA, and the resulting large plasmid may be purified and used in vivo or ex vivo human gene therapy.
Methods of the invention described herein for preparing pharmaceutical grade plasmid DNA that is highly pure and intact are advantageous over prior art methods in that the methods described herein are scalable and thus amenable to scale-up to large-scale manufacture. Such methods do not depend upon purification techniques which utilize toxic organic extractants or animal-derived proteins such as lysozyme and proteinases. Nor do methods of the invention require the use of mutagenic reagents such as ethidium bromide for plasmid DNA isolation. Nor do the methods of the invention require the use of large volumes of flammable solvents such as ethanol or propan-2-ol.